The Search for the CureThe Search for the Cure

St. Josephs Cancer Research

Hill Park Secondary School, The Co-Op & Science Departments and Mr. Watts

wish to thank

Dr. D. Tang

for allowing Brandon Tate to do a Co-Op placement in his lab (McMaster University/St. Joseph’s Hospital)

and

Andrew,

a PhD student in Dr. Tang’s lab who both graciously gave of their time to supervise Brandon’s work on a daily basis.

Acknowledgement

From my placement I expected to learn many new lab From my placement I expected to learn many new lab protocols and generally immerse myself amongst protocols and generally immerse myself amongst

professionals in the laboratory setting.professionals in the laboratory setting.

What I Expected to Learn From This Placement

Oncogene – a.k.a. the accelerator, they are genes that cause rapid cell division

Medium – a nutrient rich fluid that includes an antibiotic to ensure cell growth and specificity

PIN – prostatic intraepithelial neoplasia

Tumor Suppressor – a.k.a. the brakes, they are genes that slow cell division

Proliferation – rapid cell division

Antibody – a protein that is formed by the immune system that binds to foreign material

General Terms

Cancer is a mutation in DNA that allows cells to proliferate with no bounds

Approximately 92% of prostate cancer occurs in men over the age of 55

EVERY man will have some degree of prostate cancer but not all will be life threatening

A tumor may grow without any nutrition and minimal air

A tumor secretes a certain substance that creates its own blood vessel network which attaches to the persons circulatory system

Facts About Cancer

How it works A plasmid (circular DNA) is

cut at specific sites by restriction enzymes

It is then linear and accepts other genes easier

The gene to be transferred is cut out of another plasmid by restriction enzymes which are hopefully similar to the ones used to cut the other plasmid

If they are not the same restriction enzymes, alternate steps must be performed to insert the target gene into the plasmid

We then confirm the cuts by running a DNA gel

Through many more processes we purify the newly formed plasmid and introduce it into a cell

An antibiotic resistant gene is also included in the cell to guarantee that only cells with our plasmid are the ones we are studying

Subcloning

Subcloning

Use of subcloning Subcloning is used to control the expression of a gene

Some websites for illustrated learning of subcloning http://www.biology.ualberta.ca/facilities/multimedia/uploa

ds/microbiology/ecoli.swf http://www.101science.com/Video.htm

1: 50L Reactions2: 6L Sodium Acetate3: 120L 100% Ethanol4: 176L in -20 for 5 min

5: Spin +4-13000 RPM for 10 min6: Remove supernatant7: Wash 500L 70 % Ethanol8: Spin +4-13000 RPM for 10 min9: Remove Supernatant10: Vacuum centrifuge to dry11: 43L H2O to solve precipitate DNA (room temp. 5

min)12: 5L pfu buffer13: 10L DNTP14: 1L pfu15: 50L => PCR machine for 30 min

16: add 50L H2O17: 100L chloroform18: mix 200L hand 1 min19: mix top speed 1min in centrifuge20: keep supernatant21: repeat steps 5-1022: 43L H2O23: 5L BAMH1, buffer III24: 2L BAMH125: 50L +37 incubator overnight

Sample Procedure

ImmunohistochemistryImmunohistochemistry is a process that

involves visible dyes and antibodies

Immunofluorescence is a process that involves invisible dyes and antibodies

The light emitted by the tissue may only be seen under different wavelengths of fluorescent light

At this lab we currently have three different stains which fluoresce blue, green, and red when viewed under the microscope

The blue stain is typically used for marking the nucleus The other two differ between experiments Tissue samples actually “auto-fluoresce” which generally means

that they emit their own fluorescent light without actually being stained

This “auto-fluorescence” is deemed background and is a formidable opponent for any researcher

The goal of staining slides with immunofluorescent dyes is to target enzymes at specific sites in cells

In order to achieve this we must obtain signal over background

Immunofluorescence

Auto-fluorescence Chart

Pat.# % Intensity % of Slide IHC or IF7 80 90 IHC12 50-60 70 IHC8 Ca/N 95-100 / n/a 80 / n/a IHC / n/a (due to lack of glands)5 n/a n/a n/a (due to lack of glands)9 Ca/N 0-70 / 0-20 50 / <10 IF / IF13 0-10 0 IF(small amount of glands)34 <10 <5 IF11 Ca/N 95-100 / 0-30 70 / 25 IHC / IF2 25 80 IHC3 Ca/N 0-100(varied) / 40 20 / 60 IF / IHC10 Ca/N 85 / 50-75 20 / 60 IF / IHCTotal of 16 slides 7=>IF / 9=>IHC

All slides suitable for IF should be previewed before staining to see areas of high AF

Immunohistochemistry Protocol- (Part 1) Deparaffinize and rehydrate 3 x 10min in xylene; 2 x 2min in 100% EtOH, 2

x 2min in 70% EtOH. Rinse slides gently with ddH20 squirt bottle (b

e careful not to rinse directly on section). Wash with ddH20 in Coplin jar for 2 x 2min on shaker at 50rpm.

Immunofluorescence Protocol

Quenching Endogenous Peroxidase

Wipe off excess H2OCover section with 3% H2O2 for 10min(This step is to essentially eliminate all of

the peroxidase that is endogenous to the tissue thereby stopping non-specific binding.)

Wash with 1 x PBS in squirt bottle and then in coplin jar for 2 X 2 min on shaker at 50 rpm

Antigen retrieval

Pre-heat Antigen Retrieval Buffer in food steamer for at least 10-15min.

Incubate slides in pre-heated Antigen Retrieval Buffer for 20min.(this process breaks aldehyde crosslinks which were formed at the time of preservation by using heat and a citrate buffer)

Remove lid from food steamer and let slides sit for 10 min.

Remove entire slide box (with buffer) and let sit at room temperature on bench for 20min.

Wash slides with I x PBS for 3 x 5 min in Coplin jar on shaker.

Blocking and primary antibody addition

Gently dry slides and put in humidity chamber. Blocking buffer: 3% normal goat serum, 3%BSA with

I x PBS Incubate slides for I hr at room temperature with blo

cking buffer:(blocks non-specific binding sites giving us a more accurate result)

Wipe and shake off excess blocking buffer. Add primary antibody diluted in blocking buffer and i

ncubate overnight at 4°C.(primary antibody binds to antigens on target)

Immunohistochemistry Protocol- (Part 2)

Wash slides 3 x 5 min PBS Add biotinylated secondary antibody for 1 hr

(secondary attaches to primary antibody) Prepare ABC (Avidin-Biotin Complex) reagent let sit for 30 min Wash slides 3 x 5 min PBS Add ABC reagent, incubate for 1 hr

(The avidin and biotinylated peroxidase provide an amplification of the signal )

Wash slides 3 x 5 min PBS Add DAB (Diaminobenzidine) solution, times range from 2-10 min

(DAB forms a precipitate when reacted with peroxidase giving us our brown signal)

Wash slides with ddH2O for 30 sec and incubate in coplin jar for 5 min Counterstain with haemotoxylin for as short as possible Wash slides with ddH2O for 30 sec and incubate in coplin jar for 5 min Clear and dehydrate slides (2 x 3 min 70% EtOH, 2 x 3 min 100% EtOH, 2 x 3 min

xylenes Coverslip with cytoseal mounting medium

Immunofluorescence Protocol- (Part 2)

Wash slides 3 x 5min in I x TBST Add appropriate fluorescent secondary antibody (1:50) to each

section. Small volume: (20L per I mL blocking buffer), Incubate in chamber for 1 hr at room temperature

Wash slides for 3 x 5min in l x TBST Clear and dehydrate slides

(opposite of depariffinize and rehydrate) Coverslip with vectashield mounting medium (contains the DA

PI which stains the nucleus blue)

Immunofluorescence follows the same immunohistochemistry protocol. The only differences are immunofluorescence does not have a quenching endogenous peroxidase step and also instead of PBS we wash the slides with TBST, a detergent.

ConclusionsIn my extensive immunhistochemistry staining and analyzing of slides for CDK 11 and FADD we have learned that CDK 11 localizes

to the cytoplasm in prostate carcinoma as compared to a more nuclear localization in a normal prostate gland. FADD co-localizes with the CDK 11 pattern in prostate samples. This result suggests that the kinase, CDK 11, and the adapter FADD, work together in

the prostate.

In my analysis of BCL2 ectopically expressing MCF7 (BCL2 null), BCL2 correlated with P-ATM, P-P53, and P-CHK2. This indicates

activation of a DNA damage / tumour surveillance pathway.

Included in my conclusions is a chart which, at the moment, includes the % of PIN, Ca, and N glands on a slide.

P/N CDK11/FADD Ca/N Intensity (0=none, 5=highest) %PIN %Ca %N %Total(check)Ca 47 31 22 100NCa 30 54 16 100NCa 52 18 30 100N 19 24 57 100Ca 47 20 33 100N 35 30 35 100Ca 28 38 34 100N 32 24 44 100Ca 28 35 37 100N 49 28 23 100Ca 37 28 35 100N 69 22 9 100Ca 41 37 22 100N 51 42 7 100Ca 43 20 37 100NCa 30 14 56 100NCa 54 21 25 100N N/A N/A N/ACa 51 33 16 100N N/A N/A N/ACa 64 16 20 100N 25 12 63 100Ca 58 20 22 100N 23 12 65 100Ca 21 68 11 100N 48 37 15 100Ca 10 85 5 100N 38 45 17 100Ca 32 60 8 100N 72 23 5 100Ca 19 67 14 100N 54 37 9 100Ca 67 12 21 100N 73 104 23Ca 71 6 23 100NCa 12 65 23 100NCa 17 59 24 100NCa 19 77 4 100N 45 49 6 100CaNCa 46 31 23 100NCa 31 50 19 100NCa 28 41 31 100NCa 32 48 20 100NCa 27 50 23 100N 27 5 68 100Ca 33 55 12 100N 25 9 66 100Ca 25 60 15 100NCa 18 48 34 100NCa 26 61 13 100N 11 29 60 100Ca 27 58 15 100N 42 42 16 100CaNCa

FADD

CDK11

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FADD

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CDK11

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FADD

CDK11

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CDK11

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CDK11

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CDK11

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CDK11

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CDK11

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CDK11

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CDK11

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CDK11

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CDK11

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CDK11

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Immunohistochemistry PicturesImmunohistochemistry Pictures

Thin, elongated basal cell layer

Prominent luminal cell layer

CDK11 - NormalCDK11 - Normal

FADD - NormalFADD - Normal

Degradation of basal cell layer

Cells proliferating into lumen

CDK11 - PINCDK11 - PIN

FADD - PINFADD - PIN

Non-existent basal cell layer

Small glands

Loss of contact inhibition

CDK11 - CarcinomaCDK11 - Carcinoma

FADD - CarcinomaFADD - Carcinoma

Immunofluorescence Immunofluorescence FiguresFigures

Bcl-2 P53 Merge DAP1 Merge + DAP1

Bcl-2 CHK2 Merge DAP1 Merge + DAP1

Bcl-2 ATM Merge DAP1 Merge + DAP1

What I LearnedWhat I Learned

From my experiences in the lab I have learned an From my experiences in the lab I have learned an abundance of techniques and skills that without abundance of techniques and skills that without

this placement I would not have learned for this placement I would not have learned for many years to come. Everything I learned in the many years to come. Everything I learned in the

lab (immunofluorescence, lab (immunofluorescence, immunohistochemistry, subcloning, how to put immunohistochemistry, subcloning, how to put together figures, all the abbreviations that are together figures, all the abbreviations that are

included in a research discussion, etc.) I will be included in a research discussion, etc.) I will be able to use in the coming years of university. If I able to use in the coming years of university. If I had a chance to change one thing it would be had a chance to change one thing it would be my organization, which constantly plagues my my organization, which constantly plagues my

work. work.

Thank youThank you

Thank you to everyone in the lab for all of the help Thank you to everyone in the lab for all of the help and dealing with my constant questions. and dealing with my constant questions.

A special thank you to Dr. Tang for allowing me A special thank you to Dr. Tang for allowing me this amazing opportunity to work alongside your this amazing opportunity to work alongside your gifted employees and for bringing me one step gifted employees and for bringing me one step

closer to my life goals.closer to my life goals.